动物组织中RNA/cDNA/基因组DNA检测方案(研磨机)

智能文字提取功能测试中

Homogenization CellLysisGENO/GRINDER APPLICATIONNOTE Extraction ofRNA/cDNA and Genomic DNA from Tissue withReal-Time PCR With kind permission of Christian M. Leutenegger， Ph.D.， University of California， Davis，cmluetenegger@ucdavis.edu， January 2003 Sample Collection， Preparation and Tissue Grinding Fresh samples of animal tissue were collected， trimmed to approximately 50 - 100 mg of wetweight， snap-frozen in liquid nitrogen， and stored at -80C. The animals were human，dog，cat， mouse， cow， and horse， as well as fish and clams； see Table 1. Before DNA and/or RNAextraction， the tissues were transferred frozen to a deep-well titer plate standing on a block ofdry ice. Each well contained two 4-mm stainless steel balls (SPEX SamplePrep cat. no. 2150) and500 microliters of buffer (Applied Biosystems nucleic acid purification lysis buffer). The plateswere sealed with a plastic cover and subjected to grinding in the Geno/Grinder for 2 minutes ata setting of 1000 strokes per minute. After 30 minutes at 4C， lysates were used for either gDNAextraction or total RNA extraction.Conditions were optimized for an Applied Biosystems 6700automated nucleic acid (ANA) workstation， according to the manufacturer's instructions. Thefinal amount of tissue subjected to RNA and/or DNA extractions was between 10 and 20 mg. Quality Control of Extracted RNA/cDNA and Genomic DNA UsingReal-Time Taqman° PCR To assess the quality of the extracted RNA， complementary DNA (cDNA) was synthesized usingInvitrogen products： 200 units of SuperScript III， 600 ng of random hexadeoxyribonucleotide(pd(N)6) primer (random hexameter priner)， 10 U RnaseOut (Rnase inhibitor)， and 1 mM dNTPsin a final volume of 40 pL. The reverse transcription reaction took place for 50 minutes at 50°C.After addition of 60 uL of water， the reaction was terminated by heating for 5 minutes to 95°Cand cooling on ice. The quality of the cDNA is judged according to the CT value obtained with aendogenous control TaqMan PCR system from a defined amount of tissue. Samples with valuesabove a certain threshold indicate impaired sample quality and degradation of total RNA andwarrant re-extraction of a back-up sample. To assess the quality of cDNA， we normally useTaqMan PCR systems targeting species specific glyceraldehyde-3-phosphate dehydrogenase(GAPDH) or ribosomal genes (such as 18S rRNA or ITS-2). To assess the quality of extracted genomic DNA， species specific TaqMan PCR systems weredeveloped targeting single copy genes to allow the quantitation of genomeequivalents andcell numbers. An overview of TaqMan systems used for gDNA quality control is given in Table1. The gDNA quality from a defined amount of tissue is judged according to the CT valueusing a TaqMan PCR system targeting a single copy gene. Samples with values above a certainthreshold indicate degraded DNA and warrant re-extraction of a back-up sample. GAPDHTaqMan systems can be used to target the single copy GAPDH pseudogene (Galland et al.，1990； Garcia-Meunier et al.， 1993). SPEX SamplePrepP ：： APPLICATION NOTE SP021：Extraction of RNA/cDNA andGenomic DNA from Tissue with Real-Time PCR ：： APPARATUS：Geno/Grinder@2010 ：：APPLICATION：DNA/RNA and Other Extractions SPEX SamplePrep 65 Liberty St Metuchen， NJ 08840 USA Tel：732-623-0465 Fax： 732-906-2492 E-mail： Sampleprep@spex.com www.spexsampleprep.com European Headquarters SPEX Europe ：2 Dalston Gardens Stanmore，HA7 1BQ， UK Tel：+44 (0)208 204 6656 Fax：+44 (0)208 204 6654 E-mail： spexeurope@spex.com Web： www.spexeurope.com Each real-time TaqMan PCR reaction contained 400 nM of each primer， 80 nM of the TaqManprobe and commercially available PCR mastermix (TaqMan Universal PCR Mastermix，Applied Biosystems) containing 10 mM Tris-HCI (pH 8.3)， 50 mM KCI， 5 mM MgCl2， 2.5 mMdeoxynucleotide triphosphates， 0.625 U AmpliTaq Gold DNA polymerase per reaction， 0.25U AmpErase UNG per reaction and 5 pL of the diluted cDNA sample or the gDNA in a finalvolume of 25 uL. The samples were placed in 96 well plates and amplified in an automatedfluorometer (ABI PRISM 7700 Sequence Detection System， Applied Biosystems). Amplificationconditions were 2 min at 50℃， 10 min at 95℃， 40 cycles of 15 s at 95℃ and 60 s at 60℃. References GAPDH pseudogenes： Galland，F.， M. Stefanova， V. Pirisi， and D. Birnbaum.1990. Characterization of a murineglyceraldehyde-3-phosphate dehydrogenase pseudogene. Biochimie. 72：759-62. Garcia-Meunier， P.， M. Etienne-Julan， P. Fort， M. Piechaczyk，and F. Bonhomme.1993. Concertedevolution in the GAPDH family of retrotransposed pseudogenes. Mamm.Genome. 4：695-703.Leutenegger， C.M.， B.von Rechenberg， K. Zlinsky， C. Mislin， M. Akens， J. Auer， and H. Lutz： Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embeddedin methyl methacrylate. Calcified Tissue International， 65：378-383， 1999. Leutenegger， C.M.， A.M. Alluwaimi， W.Smith， L. Perani， J.M. Cullor. Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction.Veterinary Immunology and Immunopathology. 77：275-287，2001. Leutenegger， C.M.. The real-time TaqMan PCR and applications in Veterinary Medicine.Veterinary Sciences Tomorrow， Online Journal， Jan 1， 2001. Table 1Overview of tissue samples， species and TaqMan systems used to assess quality of cDNA and gDNA SPEX SamplePrep 65Liberty St Metuchen， NJ 08840 USA Na： Not Available Tel：732-623-0465 CT Value： Cycle Threshold Value： PCR cycle at which the fluorescent intensity exceeds the threshold Fax： 732-906-2492 E-mail： Sampleprep@spex.com www.spexsampleprep.com European Headquarters SPEX Europe 2 Dalston Gardens Stanmore，HA7 1BQ， UK Tel：+44(0)208 204 6656 Fax：+44 (0)208 204 6654 E-mail：spexeurope@spex.comWeb： www.spexeurope.com Table 2 Composition of real-time fluorogenic Taqman PCR assay GAPDH IL4 IL10 IL 12 p35 IL 12 p40 IFN-g II16 2xMastermix 12.5 12.5 12.5 12.5 12.5 12.5 12.5 10x Buffer A 1x 1x 1x 1x 1x 1x 1x MgCl2(mM) 5 5 5 5 5 5 5 dATP (mM) 300 300 300 300 300 300 300 dCTP(mM) 300 300 300 300 300 300 300 dGTP (mM) 300 300 300 300 300 300 300 dUTP (mM) 300 300 300 300 300 300 300 Forward 400 400 400 400 400 400 400 primer (nM) Reverse 400 400 400 400 400 400 400 primer (nM) TaqMan 80 80 80 80 80 80 80 probe (nM) AmpliTaqGold (U) 0.75 0.75 0.75 0.75 0.75 0.75 0.75 AmpErase UNG(U) 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Template(cDNA) 10 ml 10 ml 10 ml 10 ml 10 ml 10ml 10 ml H20 Ad 25 ml Ad 25 ml Ad 25 ml Ad 25 ml Ad 25 ml Ad 25 ml Ad 25 ml Your Science is Our Passion. 10 xTaqMan Buffer A contains 500 mM KCl， 100 mM Tris-HCl， 100 mM EDTA， 600 nM， passivereference ROX， pH 8.3 at room temperature 65 Liberty St Metuchen， NJ 08840 USA Tel： 732-623-0465 Fax： 732-906-2492 E-mail： Sampleprep@spex.com www.spexsampleprep.com European Headquarters SPEX Europe 2 Dalston Gardens Stanmore，HA7 1BQ， UK Tel： +44 (0)208 204 6656 Fax： +44 (0)208 204 6654 E-mail： spexeurope@spex.com Web： www.spexeurope.com 1、样品采集、制备和组织研磨采集新鲜动物组织样本，修剪至约50-100mg湿重，在液氮中快速冷冻，并保存在-80℃低温冰箱中。样品来自于人、狗、猫、老鼠、牛、马，还有鱼和蛤（见表1所示）。在提取DNA或RNA之前，将这些冷冻后的组织，放入深孔板中，并将深孔板放置在干冰上冷冻。每个孔中放入两颗4m钢珠(SPEX货号2150) ，再加入500mL缓冲液(应用生物系统核酸纯化裂解缓冲液)。用深孔板盖板密封，在高通量组织研磨仪上以每分钟1000次的速度研磨2min。4℃环境下培养30min后，用裂解液提取gDNA或总RNA。使用高通量组织研磨仪优化后，再使用6700全自动核酸工作站进行实时PCR，提取到的RNA或DNA的组织量在10到20mg之间。 2、采用Real-Time Taqman PCR技术对提取的RNA/cDNA和基因组DNA进行质量控制为评价提取的RNA的质量，采用Invitrogen产品合成互补DNA (cDNA)：200 units of SuperScript III, 600 ng of random hexadeoxyribonucleotide (pd(N)6) primer (random hexameter priner), 10 U RnaseOut (Rnase inhibitor), and 1 mM dNTPs in a final volume of 40 L.逆转录反应在50℃下进行50min，加入60微升水，加热5min至95℃，在冰上冷却，终止反应。根据内源性对照TaqMan PCR系统从一定数量的组织中获得的CT值判断cDNA的质量。值高于一定阈值的样品表明样品质量受损，总RNA降解，需要重新提取备用样品。为了评估cDNA的质量，我们通常使用的TaqMan PCR系统，目标是物种特异性的甘油醛-3-磷酸脱氢酶(GAPDH)或核糖体基因(如18S rRNA或ITS-2)。为了评估提取的基因组DNA的质量，开发了针对单个拷贝基因的物种特异性TaqMan PCR系统，以定量基因组当量和细胞数量。表1概述了用于gDNA质量控制的TaqMan系统。使用针对单个复制基因的TaqMan PCR系统，根据CT值判断一定数量组织的gDNA质量。值高于某一阈值的样本表明DNA已经降解，需要重新提取备用样本。GAPDH TaqMan系统可以用来靶向单拷贝GAPDH伪基因。每个Real-Time TaqMan每个引物的PCR反应包含400nM，80nM的TaqMan探针和市场上买到的PCR试剂(TaqMan一般性PCR试剂：Applied Biosystems)包含10mM Tris-HCl (pH值8.3)，50 mM KCl，MgCl2 2.5mM，2.5mM三磷酸脱氧核苷酸，0.625 U AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction and 5μL of the diluted cDNA sample or the gDNA in a final volume of 25 μL.样品置于96孔板中，在自动荧光仪(ABI PRISM 7700序列检测系统，Applied Biosystems)中扩增。扩增条件为50℃时为2 min, 95℃时为10 min, 95℃时为40个15s，60℃时为60 s循环。图13、结论图2使用SPEX高通量组织研磨仪研磨动物组织，可以快速高通量的完成样品制备，且核酸提取量高、提取的核酸质量好。